Biological and genetic characterization of a newly established human external auditory canal carcinoma cell line, SCEACono2

Squamous cell carcinoma of the external auditory canal (EACSCC) is an extraordinarily rare and aggressive malignant disease. Establishment of EACSCC cell line with robust molecular characteristics is essential for the basic and translational research of EACSCC. Here, we show the newly established EACSCC cell line SCEACono2, derived from a patient with well-to-moderately differentiated EACSCC. We analyzed histologic and genetic features of SCEACono2 hiring multiple experiments, including next-generation sequencing (NGS). Immunocytochemical staining of SCEACono2 showed positivity of p53 and SCC1/2. Furthermore, SCEACono2 exhibited a unique characteristic that cytokeratin, vimentin as well as cancer stem cell markers (CD44, CD133, ALP and Oct3/4) were positive. SCEACono2 had an ability to form tumors at the temporal lesion xenograft nude mice model. NGS revealed that SCEACono2 harbored the somatic mutations of TP53 (p.G245S) and NOTCH1 (p.A465T). RNA-seq and downstream bioinformatics analysis revealed significant enrichment of genes involved in inflammation and cell adhesion in SCEACono2 compared to SCC-9 and HSC-4. STR profiling indicated no evidence of cross-contamination. In conclusion, SCEACono2 could serves as a promising and robust research resource of EACSCC in vitro and in vivo.

Squamous cell carcinoma of the external auditory canal is extraordinarily rare [1][2][3][4] .The external auditory canal is divided into bony and cartilaginous parts.Both parts are covered with cutaneous tissue, and the tympanic membrane is at the most medial part.Chronic inflammation produced especially by frequent ear-picking can be caused carcinogenesis in the skin covering the bone and cartilaginous part of the external auditory canal 5 .However, its rarity makes it difficult to set up prospective clinical studies and complicates retrospective metaanalysis, preventing the accumulation of high-quality evidence.Furthermore, the delay in establishing the cell line causes the lack of basic research evidence.So far, few reports of molecular biological studies on external auditory canal carcinoma have been reported.Almost studies have mainly used the histopathological/immunohistochemistrical method on clinical samples [6][7][8][9][10][11] .
The establishment of the stable cell line of the external auditory canal carcinoma, which can be the most crucial tool for the experiments both in vivo and in vitro, has been desired to accumulate the evidence of molecular biology study.A stable cell line of this carcinoma is mandatory to reveal the precise molecular characterization of external auditory canal squamous cell carcinoma and proceed with the molecular biological experiment both in vivo and in vitro.Now reported cell line of external auditory canal squamous cell carcinoma was established by Mario A. Hermsen's group in Spain 12 .Until now, there has not been a report on the cell line derived from the Asian population.In Japan, ear picking, also called "mimikaki", is a popular habit and a unique culture, which has been considered a risk factor for chronic inflammation leading to the carcinogenesis of the external

SCEACono2 is positive for SCC markers and cancer stem cell markers
We next validated the characteristics of SCEACono2 as a SCC-derived cell line.For this purpose, we performed immunocytochemical staining of SCC marker proteins.SCEACono2 exhibited positivity for p53 and SCCA1/2 (Fig. 2A).Furthermore, both cytokeratin and vimentin were positive, suggesting a hybrid state of epithelial and mesenchymal characteristics (Fig. 2B).In order to examine stemness-like features of SCEACono2 cells, the presence of cancer stem cell markers (CD44, CD133, Oct3/4, ALP) was examined [14][15][16][17] .SCEACono2 cells showed positive for all of these cancer stem cell markers (Fig. 3A-D).

Chromosomal instability of SCEACono2 cell line
The complicated karyotype and abnormal chromosome number of the SCEACono2 cells were revealed by chromosome analysis (Fig. 4A).Chromosome number analysis was performed on 20 cells.The number of chromosomes varied from 74 to 81, with 81 being the most common (6 cells) (Fig. 4B).Among these cells, karyotyping analysis was also performed on 8 cells that could be analyzed.Eight cells showed abnormal karyotypes, indicating chromosomal instability.We observed the five most common chromosomal aberrations in these cells as follows; (1) A chromosome with an additional fragment of unknown origin on the short arm of chromosome 9. (2) A chromosome with an additional fragment of unknown origin on the short arm of chromosome 14.
(3) A chromosome with an additional fragment of unknown origin on the long arm of chromosome 18.
The karyograms of each cell are shown in Supplementary Fig. 1.To rule out the possibility of cross-contaminations with available cell lines, we performed STR analysis of SCEA-Cono2.We have extracted the genomic DNA from the SCEACono2 cells after 100 times passaging.Although the STR profile of the cultured SCEACono2 cells did not exactly match that of the original patient tumor sample, their STR similarity was 0.96, indicating that the cultured SCEACono2 highly resembles the original tumor cells.
(Fig. 5 and Table 1) We, therefore, considered these cells to be identical cell strains.In addition, we confirmed that there were no other identical cell lines in the Japanese Collection of Research Bioresources (JCRB).

Tumorigenicity in Nude mice
We next examined the tumorigenic capacity of SCEACono2 in vivo.One week after the injection of SCEACono2 cells at the right temporal region in nude mice, subcutaneous tumor size was macroscopically increased, and we resected the tumors 16 days after inoculation.(Fig. 6A, B) We examined various injection sites to validate tumorigenicity of SCEACono2, including subcutaneous groin, knee, flank, and temporal sites.Importantly,  www.nature.com/scientificreports/ the tumor formation rate of SCEACono2 exhibited 100% (n = 5), indicating that this cell line is suitable for cell line-derived xenograft models.Immunohistochemical staining showed that the xenografted tumor tissues were positive for the proliferation marker Ki 67 and p53 (Fig. 6C-E).

Whole exome sequencing (WES)
To clarify the genetic alterations of SCEACono2, we performed WES on genomic DNA derived from the cultured SCEACono2 cells, the original tumor tissues and the patient-matched PBMC as the non-cancerous control.The sequencing data had an average sequencing depth of 126.2).Among these mutations, TP53 was also found as a significant mutation in the primary tumor.Although other mutations were excluded upon filtering, we confirmed that all of them were included in the primary tumor sample with low variant read frequencies (data not shown), suggesting that the cells harboring these mutations were selected among the heterogeneous cancer cell population.Taken together, SCEACono2 resembles genetic characteristics of the primary tumor.

Transcriptomic characteristics of SCEACono2
Finally, we performed RNA-seq and downstream bioinformatics analysis to explore the gene expression signature of SCEACono2 cells.Publicly available HNSCC cell lines HSC-4 and SCC-9 were subjected to this analysis www.nature.com/scientificreports/simultaneously in order to compare their transcriptomic characteristics.Principal component analysis showed the clear separation of these three cell lines in transcriptomic levels, suggesting the unique transcriptional programs activated in SCEACono2 compared to HNSCC cell lines (Fig. 7A).We identified 1864 and 2077 upregulated genes in SCEACono2 compared to HSC-4 and SCC-9, respectively (Fig. 7B, C).In order to identify a unique transcriptional signature characterizing SCEACono2, we have extracted overlapped genes among these upregulated genes.829 genes (27%) were found to be overlapped, whereas 1035 (33%) and 1248 (40%) genes were upregulated only in the comparison to HSC-4 and SCC-9 cell, respectively (Fig. 7D).Gene ontology analysis showed significant enrichment of genes involved in the regulation of MAPK cascade (GO: 0043408), regulation of cell adhesion (GO: 0030155), response to cytokine (GO: 0034097) and inflammatory response (GO: 0006954) in these overlapping upregulated genes.(Fig. 7E).

Discussion
Squamous cell carcinomas of the head and neck include the tongue, pharyngeal, laryngeal, and sinonasal carcinomas.The development of treatment strategies for head and neck squamous cell carcinoma has been supported by building evidence from clinical and basic research.Temporal bone squamous cell carcinoma is included as a type of temporal bone squamous cell carcinoma.However, temporal bone squamous cell carcinoma of the external auditory canal and middle ear is extremely rare compared to other head and neck SCC, and the frequency of this pathogen has been reported to account for < 0.2% of head and neck squamous cell carcinoma 1,4,18 .Due to its rarity, the accumulation of molecular biological evidence and establishing of treatment guidelines have been delayed.The immortalized cell lines for each pathogen have played a major role in establishing molecular biological evidence, elucidating molecular biological mechanisms of carcinogenesis and metastasis, or verifying therapeutic resistance and radiosensitivity.The establishment of cell lines for squamous cell carcinoma of the external auditory canal has lagged far behind that of other head and neck SCC.We have previously reported a method for creating a primary culture of cancer cells isolated from patient cancer tissue.Using this method, we have established a new cell line of squamous cell carcinoma of the external auditory canal.Therefore, in this report, we verified and reported the molecular and biological characteristics of the new cell line.
According to the Cellosaurus (https:// web.expasy.org/ cello saurus/), excluding derivative cell lines, only one cell line, CAE606, is available as a cell line derived from the external auditory canal squamous cell carcinoma so far 12 .As we have previously reported, maintaining cancer cells isolated from squamous cell carcinoma of the external auditory canal is still challenging; the rarity of the disease and the difficulties in maintenance of the primary cells may explain why the establishment of immortalized cells from this disease has rarely been reported.Furthermore, the process of cell line establishment from fresh tumor tissues is energy-and time-consuming, and difficult due to contamination, out-growth of cancer-associated fibroblasts and cellular senescence, which causes the low success rate of establishment 19 .In this study, we tried to establish the new cell line from external auditory carcinoma based on the method that our group had reported previously 13 .We did not use either SV40 T-antigen, HPV E6/E7 or telomerase reverse transcriptase to immortalize cells [20][21][22][23][24][25] .During the primary cell culture, we could obtain a colony with enough mutations of the intrinsic gene to result in long-term survival, and our newly established cell line without the transfer of exogenous genes will be more suitable for studying EAC-SCC 26 .
The shape of SCEACono2 cells was quite similar to the cobblestone-like epithelioid morphology.The population doubling time was about 24 h, and this proliferative capacity can be comparable to other cells, including the Hela and SCC-9, commonly used in scientific research.Our newly established cell line harbors the typical markers of squamous cell carcinoma, including the SCCA1/2 and p53.Furthermore, this cell line expresses both vimentin and cytokeratin.This is implicated in the epithelial mesenchymal transition, associated with invasive behavior, poor differentiation, and poor prognosis.Several cancer stem cell markers, CD44, CD133, and Oct3/4, were positive, and ALP was partially positive [14][15][16][17][27][28][29] . These indings mean that this cell line has the characteristic of the stem cell partially.The SCEACono2 cells also displayed chromosomal instability, including the changes in chromosome number and the generation of hybrid DNA, which is related to the promotion of tumorigenesis and development [30][31][32][33] .
We confirmed that this new cell line could be a tool for scientific research in vivo.Injection of the newly derived cells under the temporal skin of a nude mouse resulted in tumor formation.The immunohistochemistry staining of xenograft shows the positive of Ki 67 and p53.This cell-line-derived xenograft could be used to elucidate the tumor's anatomical metastatic mechanism and select therapeutic agents for EACSCC.In this study, we analyzed the transcriptome of the newly established cell line with RNA sequencing compared to two cell lines of tongue squamous cell carcinoma: HSC-4 and SCC-9.We found that the genes related to inflammation and cell adhesion are more expressed in the newly established cell line than the other two.Furthermore, newly established cell line harbor several somatic mutations: FREM(p.S62R), GLIS3(p.S24R), COLI5A1(p.V1265E), NOTCH1(p.A465T), FSCB(p.A310V), INF2(p.R87H), and TP53(p.G245S).The somatic mutation of TP53 is the most frequently mutated tumor suppressor gene in human squamous cell carcinoma 34 .Furthermore, The somatic mutation of NOTCH1 is also commonly found in human squamous cell carcinoma 35 .These results suggest that the major drive mutations of EACSCC are similar to those of other SCC.In addition to these somatic mutations, missense mutations in FREM3, GLIS3, COLI5A1, FSCB, and INF2 are identified.However, the molecular significance of these missense mutations in EACSCC remains unclear.The previously established cell line, CAE606, harbors inactivating mutation in CDKN2A and TP53 and high-level amplification of CCND1, affecting the cell cycle pathway.Our cell line also harbors the major drive mutations of EACSCC and has the transcriptome profiling related to inflammation and cell adhesion.These analyses with next-generation analysis suggested that this newly established cell line with these genetic backgrounds is an appropriate cell line for EACSCC research.
In conclusion, we established new cell lines derived from human squamous cell carcinoma of the external auditory meatus and named SCEACono2.This newly established cell line could be a useful tool for the pathological analysis of EACSCC to provide further insight into the mechanism of tumorigenesis, metastasis, and selection of the antitumor-drug.

Ethic statement
The Clinical Research Ethics Review Committee of Kyushu University Hospital approved the study (permit no.29-43, 30-268, and 700-00).Written informed consent for the current research project was obtained before the tumor tissue, and a blood sample were harvested.This study was also conducted according to the principles of the Declaration of Helsinki.The animal experiment was also reviewed and approved by the Committee of.Ethics of Animal Experiments, Kyushu University Graduate School of Medical Sciences.The animals were treated according to the "Guidelines for Proper Conduct of Animal Experiments" (Science Council of Japan).The study was carried out in compliance with the ARRIVE guidelines.

Immunofluorescence and alkaline phosphatase staining
SCEACono2 cell for immunostaining was prepared as follows.SCEACono2 suspension for staining the Vimentin, Cytokeratin, p53, SCCA1/2, CD133, Oct3/4, and CD44 were seeded on eight-chamber slides and allowed to attach overnight.The cultures were fixed with methanol: acetone 1:1 (v/v) for 20 min at − 20 °C.These preparations for cytoplasmic antigen staining were additionally permeabilized with PBS containing 0.5% Triton X-100 and 0.05% NaN3 for 10 min at r.t.Following fixation, for cell surface marker staining, preparations were incubated with anti-CD44-PE or anti-CD133-APC for 1 h at r.t.For cytoplasmic marker staining, samples were incubated with anti-pan-Cytokeratin-Alexa Fluor® 488 and Vimentin-PE at r.t. for 1 h and with anti-p53-Alexa Fluor® 488 and anti-SCCA1/2 (mouse IgG) at 4 °C overnight.For SCCA1/2 and Oct3/4 antigens, the samples were stained with goat anti-mouse IgG (H + L) highly cross-adsorbed secondary antibody conjugated Alexa Fluor® 555 after the primary antibody reaction.Slides were then mounted with DAPI Fluoromount-G® (SouthernBiotech, Birmingham, AL) for nuclear staining.Unfixed cytospin preparations were tested with the Vector® Blue Alkaline Phosphatase Substrate kit (Vector Labs, Burlingame, CA) to check for alkaline phosphatase (ALP) activity.

Chromosome analysis: chromosomes of SCEACono2 cell
After 48 h of incubation in a culture medium, Semiconfluent and actively dividing cells were incubated in 0.5 μg/ ml Colcemid for 20 min, and cells were harvested.Harvested cells were pelleted out and treated with hypotonic solution (0.075 M KCL) for 10 min before fixation in methanol-acetic acid (3:1).The suspension of fixed cells was dropped onto microscope slides.Chromosomes of SCEACono2 cells were prepared using a standard air-drying method, metaphase spreads were counted to determine the modal number, and the G-banding technique analyzed karyotypes.The metaphase chromosome spreads slide was evaluated using Ikaros software (MetaSystems, Altussheim, Germany).

Cell-line-derived xenograft
In vivo, the tumorigenicity of the SCEACono2 cells was assessed by the ability to form tumors subcutaneously in Nude mice.SCEACono2 cells were harvested and re-suspended to prepare a cell suspension of 5 × 10 7 cells/ ml.BALB/c nude mice (5 weeks, body weight 16-18 g) were subcutaneously injected in a 0.1 ml suspension in each region.When the apparent tumor formation was macroscopically confirmed, the tumor was separated from the mice and fixed in 4% formaldehyde for pathological examination.The tumorigenesis experiment was repeated using 3 Nude mice.BALB/c nude mice were purchased from The Jackson Laboratory Japan, Inc.The xenograft tumors were examined with immunohistochemistry measure.Following deparaffinization, hydration,

Figure 4 .
Figure 4. Chromosomal numbers of SCEACono2 cells.(A) The photograph represents Giemsa-stained metaphase chromosomes from SCEACono2 cells.(B) Chromosome number analysis was performed on 20 cells.The number of chromosomes varied from 74 to 81, with 81 chromosomes being the most common (6 cells).

Figure 5 .
Figure 5. Short tandem repeat (STR) profiles.Short tandem repeat values of cell line and tumor tissue for fifteen gene loci and amelogenin (X and Y chromosomes).The evaluation value between the cell culture and host tumor tissue was 0.96, which was high enough that the STR profile of the cell line was the same as those of the host tumor sample.

Figure 6 .
Figure 6.Tumorigenicity in BALB/c mice and histological analysis of the Cell line-derived Xenograft tumors in Nude mice.(A, B) Subcutaneous tumor mass (black arrow in A and red circle in B) in BALB/c mice which inoculated with 5 × 10 6 cells at 14 days post-injection.(C) Hematoxylin and Eosin (H&E) stain of the paraffinembedded tumor sample confirmed the tumor formation of the differentiated squamous cell carcinoma.(D, E) Immunohistochemistry (IHC) analysis of SCEACono2 xenografts.Human Ki 67 and p53 were detected by IHC.The slide IHC image under low magnification and representative IHC image under high magnification of p53 and Ki 67 stain were shown on the left and right, respectively.

Figure 7 .
Figure 7.Comparison of gene expression among the newly established cell line, HSC-4, and SCC-9.(A) Principal component analysis showed the clear separation of these three cell lines in transcriptomic levels.(B) Volcano plot showing gene expression (FDR < 0.05; Log2FC > 1) altered (highlighted in red color) on the comparison between the SCEACono2 cell and HSC-4 cell.(C) Volcano plot showing gene expression (FDR < 0.05; Log2FC > 1) altered (highlighted in red color) on the comparison between the SCEACono2 cell and SCC-9 cell.(D) Venn diagram showing commonly upregulated genes in both comparisons of gene expression between the SCEACono2 cell line and HSC-4 and between the SCEACono2 cell line and SCC-9.Genes highlighted in pink are observed to be commonly upregulated in both comparisons.(E) Gene ontology (GO) enrichment analysis of upregulated genes.[− log10(p value)] of the corresponding biological process is shown.A dark orange bar indicates a small p value (p value < 0.1).

Table 2 .
Newly established cell line harbor several somatic mutations, missense mutations.